Despite more than a century of research since the discovery of Mycobacterium tuberculosis, the aetiological agent of tuberculosis, this disease remains one of the major causes of human morbidity and mortality. There are an estimated 3 million deaths annually attributable to tuberculosis (sec, D. Snider, Rev. Inf. Dis., S335 (1989)), and although the majority of these are in developing countries, the disease is assuming renewed importance in the W. due to the increasing number of homeless people and the impact the AIDS epidemic (see, R. E. Chaisson et al., Am. Res. Resp. Dis., 23, 56 (1987); D. E. Snider, Jr. et al., New Engl. J. Med, 326, 703 (1992); M. A. Fischl et al., Ann. Int. Med., 117, 177 (1992) and ibid. at 184.
Isonicotinic acid hydrazide or isoniazid (INH) has been used in the treatment of tuberculosis for the last forty years due to its exquisite potency against the members of the "tuberculosis" groups--Mycobacterium tuberculosis, M. bovis and M. africanum (G. Middlebrook, Am. Rev. Tuberc., 69, 471(1952) and J. Youatt, Am. Rev. Resp. Dis., 99, 729 (1969)). Neither the precise target of the drug, nor its made of action are known, but INH treatment results in the perturbation of several metabolic pathways of the bacterium. However, shortly after its introduction, INH-resistant isolates of Mycobacterium tuberculosis emerged. See M. L. Pearson et al., Ann. Int. Med., 117, 191(1992) and S. W. Dooley et al., Ann. Int. Med., 117, 257 (1992).
Several investigators have associated the toxicity of INH for mycobacteria with endogenous catalase activity. See, for example, "Isonicotinic acid hydrazide," in F. E. Hahn, Mechanism of Action of Antibacterial Agents, Springer-Verlag (1979) at pages 98-119. This relationship was strengthened by a recent report by Ying Zhang and colleagues in Nature, 358, 591 (1992) which described the restoration of INH susceptibility in an INH resistant Mycobacterium smegmatis strain after transformation using the catalase-peroxide (katG) gene from an INH sensitive M. tuberculosis strain. In a follow-up study, Zhang and colleagues in Molec. Microbiol., 8, 521 (1993) demonstrated the restoration of INH susceptibility in INH resistant M. tuberculosis strains after transformation by the functional katG gene. As reported by B. Heym et al., J. Bacieriol., 175, 4255 (1993), the katG gene encodes for a 80,000-dalton protein.
A conclusive diagnosis of tuberculosis depends on the isolation and identification of the etiologic agent, Mycobacterium tuberculosis, which generally requires 3-8 weeks. Design of an appropriate therapeutic regimen depends on the results of subsequent antituberculosis susceptibility testing by the agar dilution method and produces additional delays of 3-6 weeks (Roberts et al., "Mycobacerium" in Manual of Clincial Microbiology, 5th Ed.; A. Balows et al., Eds.; American Society for Microbiology: Washington; pp. 304-399 (1991). Identifcaiton and drug resistance testing can now also be accomplished more quickly by using the BACTEC radiometric method. (Tenover et al., J. Clin. Microbiol., 31 767-779 (1993) and Huebner et al., J. Clin. Microbiol., 31, 771-775 (1993). Acid fast bacilli are detected in the BACTEC bottle, and and identification is made using a nucleic acid hybridization technique on the BACTEC-derived growth. Drug susceptibility testing is then conducted using the same BACTEC growth to inoculate fresh BACTEC bottles containing various antituberculosis drugs. This procedure reduces the time needed to generate a complete analysis, but the total time required to report susceptibility results for MTB is typically in excess of 20 days. The need to minimize the transmission of newly identified drug resistant strains of MTB requires the development of much more rapid identification procedures.
The rapid detection of M. tuberculosis directly from clinical samples has been possible recently by virtue of the availability of polymerase chain reaction (PCR) and the recognition of diagnostic sequences amplified by the appropriate primers. The ability to conduct PCR analyses depends on having a high enough gene or gene product concentration so that the molecular tools work efficiently even when the organism numbers are low. Thus, the most efficient molecular assays used to detect M. tuberculosis depend on the IS6110 insertion sequence (about 10 copies) or the 16S ribosomal RNA (thousands of copies). See, respectively, K. D. Eisenach et al., J. Infect. Dis., 61, 997 (1990) and N. Miller et al., Abstracts ASM, Atlanta, Ga. (1993) at page 177. However, these methods do not provide any information regarding the drug-resistance phenotype of the M. tuberculosis strain.
Recently, B. Heym et al. (PCT WO 93122454) disclose the use of polymerase chain reaction to amplify portions of the katG gene of putative resistant strains. The PCR products were evaluated by single-strand conformation polymorphism (SSCP) analysis, wherein abnormal strand motility on a gel is associated with mutational events in the gene. For example, in five strains, a single base difference was found in a 200 bp sequence, a G to T transversion at position 3360. This difference would result in the substitution of Arg-461 by Leu. However, carrying out SSCP on a given clinical sample can be a laborious procedure that requires sequencing to confirm whether mutations or deletions predictive of drug resistance are in fact present in the target gene.
There is a continuing need in the art to develop a simple test permitting the rapid identification of M. tuberculosis and its drug-resistance phenotype.